The env gene of HIV-1 rapidly evolves. One important determinant of envevolution is selection for altered co-receptor use. Change in co-receptor use represents an important change in the biology of the virus that is correlated with accelerated disease progression. The PI has recently applied a new technology, heteroduplex tracking analysis (HTA), to study alterations in the V3 region of HIV-1 env. This region of env is the major co- receptor use determinant. The Pi proposes to use this assay to study the evolution of in V3 in patients who are at risk for the development of V3 evolutionary variants, who have undergone dramatic losses of CD4+ T- cells, and who have undergone antiretroviral therapy. They will also use this assay to study the relative growth rates of V3 variants in several model systems. They will use an experimental protocol based on strong genetic selection to explore the determinants of co-receptor use and analyze interactions with co-receptors. Finally, they will use HTA to follow specific events in other variable regions of HIV-1 env. These efforts represent a comprehensive approach to understanding the dynamic behavior of env sequence evolution and the forces driving this evolution.